Novel gene expressed in prostate cancer

ABSTRACT

A novel testis-specific gene expressed in human prostate cancer, designated 22P4F11, is described. Analysis of 22P4F11 mRNA expression in normal prostate, prostate tumor xenografts, and a variety of normal tissues indicates that the expression of this gene is testis specific in normal tissues. The 22P4F11 gene is also expressed in human prostate tumors, in some cases at high levels. A full length cDNA encoding 22P4F11 is provided. The 22P4F11 transcript and/or protein may represent a useful diagnostic marker and/or therapeutic target for prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/334,561filed 30 Dec. 2002 which is a divisional of U.S. application Ser. No.09/410,132 filed 30 Sep. 1999 which issued as U.S. Pat. No. 6,509,458which claims benefit under 35 USC § 119(e) to U.S. Ser. No. 60/146,584filed 28 Jul. 1999 and of U.S. Ser. No. 60/102,572 filed 30 Sep. 1998.The contents of these documents are incorporated herein by reference.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedproteins, termed 22P4F11, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers which express22P4F11, particularly including prostate cancers.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) 5ll582003lllSeqlist.txt Sep. 12,2008 9,864 bytes

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

Generally speaking, the fundamental problem in the management of thedeadliest cancers is the lack of effective and non-toxic systemictherapies. Molecular medicine promises to redefine the ways in whichthese cancers are managed. Unquestionably, there is an intensiveworldwide effort aimed at the development of novel molecular approachesto cancer diagnosis and treatment. For example, there is a greatinterest in identifying truly tumor-specific genes and proteins thatcould be used as diagnostic and prognostic markers and/or therapeutictargets or agents. Research efforts in these areas are encouraging, andthe increasing availability of useful molecular technologies hasaccelerated the acquisition of meaningful knowledge about cancer.Nevertheless, progress is slow and generally uneven.

As discussed below, the management of prostate cancer serves as a goodexample of the limited extent to which molecular biology has translatedinto real progress in the clinic. With limited exceptions, the situationis more or less the same for the other major carcinomas mentioned above.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy continue to be the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the management of this disease. Although the serum PSAassay has been a very useful tool, its specificity and general utilityis widely regarded as lacking in several important respects.

Most prostate cancers initially occur in the peripheral zone of theprostate gland, away from the urethra. Tumors within this zone may notproduce any symptoms and, as a result, most men with early-stageprostate cancer will not present clinical symptoms of the disease untilsignificant progression has occurred. Tumor progression into thetransition zone of the prostate may lead to urethral obstruction, thusproducing the first symptoms of the disease. However, these clinicalsymptoms are indistinguishable from the common non-malignant conditionof benign prostatic hyperplasia (BPH). Early detection and diagnosis ofprostate cancer currently relies on digital rectal examinations (DRE),prostate specific antigen (PSA) measurements, transrectalultrasonography (TRUS), and transrectal needle biopsy (TRNB). Atpresent, serum PSA measurement in combination with DRE represent theleading tool used to detect and diagnose prostate cancer. Both havemajor limitations which have fueled intensive research into findingbetter diagnostic markers of this disease.

Similarly, there is no available marker that can predict the emergenceof the typically fatal metastatic stage of prostate cancer. Diagnosis ofmetastatic stage is presently achieved by open surgical or laparoscopicpelvic lymphadenectomy, whole body radionuclide scans, skeletalradiography, and/or bone lesion biopsy analysis. Clearly, better imagingand other less invasive diagnostic methods offer the promise of easingthe difficulty those procedures place on a patient, as well as improvingdiagnostic accuracy and opening therapeutic options. A similar problemis the lack of an effective prognostic marker for determining whichcancers are indolent and which ones are or will be aggressive. PSA, forexample, fails to discriminate accurately between indolent andaggressive cancers. Until there are prostate tumor markers capable ofreliably identifying early-stage disease, predicting susceptibility tometastasis, and precisely imaging tumors, the management of prostatecancer will continue to be extremely difficult.

PSA is the most widely used tumor marker for screening, diagnosis, andmonitoring prostate cancer today. In particular, several immunoassaysfor the detection of serum PSA are in widespread clinical use. Recently,a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for PSAmRNA in serum has been developed. However, PSA is not a disease-specificmarker, as elevated levels of PSA are detectable in a large percentageof patients with BPH and prostatitis (25-86%) (Gao et al., 1997,Prostate 31: 264-281), as well as in other nonmalignant disorders and insome normal men, a factor which significantly limits the diagnosticspecificity of this marker. For example, elevations in serum PSA ofbetween 4 to 10 ng/ml are observed in BPH, and even higher values areobserved in prostatitis, particularly acute prostatitis. BPH is anextremely common condition in men. Further confusing the situation isthe fact that serum PSA elevations may be observed without anyindication of disease from DRE, and visa-versa. Moreover, it is nowrecognized that PSA is not prostate-specific (Gao et al., supra, forreview).

Various methods designed to improve the specificity of PSA-baseddetection have been described, such as measuring PSA density and theratio of free vs. complexed PSA. However, none of these methodologieshave been able to reproducibly distinguish benign from malignantprostate disease. In addition, PSA diagnostics have sensitivities ofbetween 57-79% (Cupp & Osterling, 1993, Mayo Clin Proc 68:297-306), andthus miss identifying prostate cancer in a significant population of menwith the disease.

There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter et al., 1996, Proc.Natl. Acad. Sci. USA 93: 749; Bzdega et al., 1997, J. Neurochem. 69:2270). However, the expression of PSM in small intestine and brain(Israeli et al., 1994, Cancer Res. 54: 1807), as well its potential rolein neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee et al., 1996, Clin Nuc Med 21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252) andprostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl.Acad. Sci. USA 95: 1735). PCTA-1, a novel galectin, is largely secretedinto the media of expressing cells and may hold promise as a diagnosticserum marker for prostate cancer (Su et al., 1996). PSCA, a GPI-linkedcell surface molecule, was cloned from LAPC-4 cDNA and is unique in thatit is expressed primarily in basal cells of normal prostate tissue andin cancer epithelia (Reiter et al., 1998). Vaccines for prostate cancerare also being actively explored with a variety of antigens, includingPSM and PSA.

SUMMARY OF THE INVENTION

The present invention relates to a novel gene expressed in prostatecancer cells, designated 22P4F11. Analysis of 22P4F11 mRNA expression innormal prostate, prostate tumor xenografts, and a variety of normaltissues indicates that the expression of this gene is testis specific innormal tissues. The 22P4F11 gene is also expressed in human prostatecancer xenograft tumors, in some cases at high levels. A full lengthcDNA encoding 22P4F11 is provided. The 22P4F11 transcript and/or proteinmay represent a useful diagnostic marker and/or therapeutic target forprostate cancer.

The invention provides polynucleotides corresponding or complementary toall or part of the 22P4F11 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding 22P4F11proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to the22P4F11 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides which hybridize to the 22P4F11 genes, mRNAs, or to22P4F11-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 22P4F11. Recombinant DNA moleculescontaining 22P4F11 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 22P4F11gene products are also provided. The invention further provides 22P4F11proteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to 22P4F11 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, antibodies labeled with a detectable marker.The invention further provides methods for detecting the presence of22P4F11 polynucleotides and proteins in various biological samples, aswell as methods for identifying cells that express 22P4F11. Theinvention further provides various therapeutic compositions andstrategies for treating cancers which express 22P4F11 such as prostatecancers, including therapies aimed at inhibiting the transcription,translation, processing or function of 22P4F11 and cancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Nucleotide and deduced amino acid sequences of a full lengthcDNA encoding the 22P4F11 gene (SEQ ID NOS: 1 and 2, respectively). Theconsensus Kozak sequence and start methionine are indicated in bold, anda putative mitochondrial signal sequence is boxed.

FIG. 1B. Nucleotide and ORF amino acid sequences of SSH-isolated 22P4F11cDNA (SEQ ID NOS: 3 and 4, respectively).

FIG. 2. RT-PCR analysis of 22P4F11 gene expression in prostate cancerxenografts, normal prostate, and other tissues and cell lines, showingexpression in prostate cancer xenografts and normal prostate atapproximately equal levels (Panel A); and showing expression in normaltissues is highest in prostate, testis, lung and liver (Panels B and C).

FIG. 3. Northern blot analysis of 22P4F11 gene expression in a varietyof normal human tissues and various prostate cancer xenografts, showingtestis-specific expression in normal tissues and in some of the prostatecancer tissues. FIGS. 3A and 3B show the results of Northern blotanalysis of samples taken from various normal human tissues usinglabeled 22P4F11-GTP3E10 cDNA as a probe. RNA samples were quantitativelynormalized with a actin probe. Expression was only detected in testis.FIG. 3C shows analysis of RNAs derived from human prostate cancerxenografts, normal prostate and a prostate cancer cell line. Strongexpression was detected in one of the LAPC-4 xenografts, and lower levelexpression was detected in the LNCaP prostate cancer cell line and insome of the other xenografts.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers which have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers which have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least 10amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C., and most preferably tostringent hybridization conditions.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative position which are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions which are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. Further details regarding amino acidsubstitutions, which are considered conservative under such criteria,are provided below.

Additional definitions are provided throughout the subsections whichfollow.

Structure and Expression of 22P4F11

As is further described in the Examples which follow, the 22P4F11 genesand proteins have been characterized using a number of analyticalapproaches. For example, analyses of nucleotide coding and amino acidsequences were conducted in order to identify potentially relatedmolecules and three distinct 22P4F11 isoforms, as well as recognizablestructural domains, topological features, and other elements within the22P4F11 mRNA and protein structures. RT-PCR and Northern blot analysesof 22P4F11 mRNA expression were also conducted.

A full length cDNA encoding the 22P4F11 gene has been isolated. Thenucleotide and deduced amino acid sequences of this cDNA are shown inFIG. 1A (SEQ ID NOS: 1 and 2, respectively). The nucleotide sequence ofthe initially isolated cDNA fragment corresponding to and identifyingthe 22P4F11 gene is provided in FIG. 1B (SEQ ID NOS: 3 and 4). Thissequence shows no homology with any known genes or ESTs, and contains anopen reading frame encoding 69 amino acids (FIG. 1B).

Expression analysis by RT-PCR shows that 22P4F11 is expressed inandrogen-dependent and androgen-independent LAPC prostate cancerxenografts and in normal prostate at approximately equal levels (FIG.2). In normal tissues, expression of the 22P4F11 gene appears somewhatrestricted, with the highest levels of expression observed in prostate,testis, lung and liver tissues (FIG. 2). Northern blot analysis using afull length 22P4F11 cDNA probe, however, shows testis-specificexpression in normal tissues as well as expression in prostate cancerxenografts, but no expression in normal prostate (FIG. 3).

22P4F11 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 22P4F11 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 22P4F11 protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a 22P4F11 gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides which hybridize to a 22P4F11 gene,mRNA, or to a 22P4F11-encoding polynucleotide (collectively, “22P4F11polynucleotides”). As used herein, the 22P4F11 gene and protein is meantto include the 22P4F11 genes and proteins specifically described hereinand the genes and proteins corresponding to other 22P4F11 proteins andstructurally similar variants of the foregoing. Such other 22P4F11proteins and variants will generally have coding sequences which arehighly homologous to the 22P4F11 coding sequence, and preferably willshare at least about 50% amino acid identity and at least about 60%amino acid homology (using BLAST criteria), more preferably sharing 70%or greater homology (using BLAST criteria). One embodiment of a 22P4F11polynucleotide has the sequence shown in FIG. 1A (SEQ ID NO:1).

A 22P4F11 polynucleotide may comprise a polynucleotide having thenucleotide sequence of human 22P4F11 as shown in FIG. 1A (SEQ ID NO:1),wherein T can also be U; a polynucleotide which encodes all or part ofthe 22P4F11 protein; a sequence complementary to the foregoing; or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide having the sequence as shown in FIG. 1 (SEQID NO:1), from nucleotide residue number 236 through nucleotide residuenumber 1466, wherein T can also be U. Another embodiment comprises apolynucleotide encoding a 22P4F11 polypeptide whose sequence is encodedby the cDNA contained in the plasmid as deposited with American TypeCulture Collection as Accession No. 98985. Another embodiment comprisesa polynucleotide which is capable of hybridizing under stringenthybridization conditions to the human 22P4F11 cDNA shown in FIG. 1A (SEQID NO:1) or to a polynucleotide fragment thereof.

Specifically contemplated are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone or including alternative bases, whether derivedfrom natural sources or synthesized. For example, antisense moleculescan be RNAs or other molecules, including peptide nucleic acids (PNAs)or non-nucleic acid molecules such as phosphorothioate derivatives, thatspecifically bind DNA or RNA in a base pair-dependent manner. A skilledartisan can readily obtain these classes of nucleic acid molecules usingthe 22P4F11 polynucleotides and polynucleotide sequences disclosedherein.

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a 22P4F11 polynucleotide in a sample and as ameans for detecting a cell expressing a 22P4F11 protein. Examples ofsuch probes include polypeptides comprising all or part of the human22P4F11 cDNA sequences shown in FIGS. 1A and 1B. Examples of primerpairs capable of specifically amplifying 22P4F11 mRNAs are alsodescribed in the Examples which follow. As will be understood by theskilled artisan, a great many different primers and probes may beprepared based on the sequences provided in herein and used effectivelyto amplify and/or detect a 22P4F11 mRNA.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides whichcorrespond or are complementary to genes other than the 22P4F11 gene orwhich encode polypeptides other than 22P4F11 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 22P4F11 polynucleotide.

The 22P4F11 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 22P4F11 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 22P4F11 polypeptides; as tools formodulating or inhibiting the expression of the 22P4F11 gene(s) and/ortranslation of the 22P4F11 transcript(s); and as therapeutic agents.

Methods for Isolating 22P4F11-Encoding Nucleic Acid Molecules

The 22P4F11 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 22P4F11 gene product(s), as well as theisolation of polynucleotides encoding 22P4F11 gene product homologues,alternatively spliced isoforms, allelic variants, and mutant forms ofthe 22P4F11 gene product. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 22P4F11 gene are wellknown (See, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition., Cold Spring Harbor Press, New York,1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,Wiley and Sons, 1995). For example, lambda phage cloning methodologiesmay be conveniently employed, using commercially available cloningsystems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing22P4F11 gene cDNAs may be identified by probing with a labeled 22P4F11cDNA or a fragment thereof. For example, in one embodiment, the 22P4F11cDNA (FIG. 1A, 1B) (SEQ ID NO:1, SEQ ID NO:2) or a portion thereof canbe synthesized and used as a probe to retrieve overlapping and fulllength cDNAs corresponding to a 22P4F11 gene. The 22P4F11 gene itselfmay be isolated by screening genomic DNA libraries, bacterial artificialchromosome libraries (BACs), yeast artificial chromosome libraries(YACs), and the like, with 22P4F11 DNA probes or primers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 22P4F11 polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YACs, BACs, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, for example, Sambrook et al, 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 22P4F11 polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 or HighFive cell). Examplesof suitable mammalian cells include various prostate cancer cell linessuch LnCaP, PC-3, DU145, LAPC-4, TsuPr1, other transfectable ortransducible prostate cancer cell lines, as well as a number ofmammalian cells routinely used for the expression of recombinantproteins (e.g., COS, CHO, 293, 293T cells). More particularly, apolynucleotide comprising the coding sequence of a 22P4F11 may be usedto generate 22P4F11 proteins or fragments thereof using any number ofhost-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of22P4F11 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 22P4F11 may be preferably expressed in severalprostate cancer and non-prostate cell lines, including for example 293,293T, rat-1, 3T3, PC-3, LNCaP and TsuPr1. The host-vector systems of theinvention are useful for the production of a 22P4F11 protein or fragmentthereof. Such host-vector systems may be employed to study thefunctional properties of 22P4F11 and 22P4F11 mutations.

Recombinant human 22P4F11 protein may be produced by mammalian cellstransfected with a construct encoding 22P4F11. In a particularembodiment described in the Examples, 293T cells are transfected with anexpression plasmid encoding 22P4F11, the 22P4F11 protein is expressed inthe 293T cells, and the recombinant 22P4F11 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-22P4F11 antibodies). In another embodiment, also described in theExamples herein, the 22P4F11 coding sequence is subcloned into theretroviral vector pSRαMSVtkneo and used to infect various mammalian celllines, including 3T3CL7, PC3 and LnCaP in order to establish 22P4F11expressing cell lines. Various other expression systems well known inthe art may also be employed. Expression constructs encoding a leaderpeptide joined in frame to the 22P4F11 coding sequence may be used forthe generation of a secreted form of recombinant 22P4F11 protein.

Proteins encoded by the 22P4F11 genes, or by fragments thereof, willhave a variety of uses, including but not limited to generatingantibodies and in methods for identifying ligands and other agents(i.e., other bHLH proteins) and cellular constituents that bind to a22P4F11 gene product. Antibodies raised against a 22P4F11 protein orfragment thereof may be useful in diagnostic and prognostic assays, andimaging methodologies in the management of human cancers characterizedby expression of 22P4F11 protein, including but not limited to cancersof the prostate. Such antibodies may be expressed intracellularly andused in methods of treating patients with such cancers. Variousimmunological assays useful for the detection of 22P4F11 proteins arecontemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting 22P4F11 expressingcells (e.g., in radioscintigraphic imaging methods). 22P4F11 proteinsmay also be particularly useful in generating cancer vaccines, asfurther described below.

22P4F11 Proteins

Another aspect of the present invention provides 22P4F11 proteins andpolypeptide fragments thereof. The 22P4F11 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined below. Fusion proteins which combineparts of different 22P4F11 proteins or fragments thereof, as well asfusion proteins of a 22P4F11 protein and a heterologous polypeptide arealso included. Such 22P4F11 proteins will be collectively referred to asthe 22P4F11 proteins, the proteins of the invention, or 22P4F11. As usedherein, the term “22P4F11 polypeptide” refers to a polypeptide fragmentor a 22P4F11 protein of at least 10 amino acids, preferably at least 15amino acids.

A specific embodiment of a 22P4F11 protein comprises a polypeptidehaving the amino acid sequence of human 22P4F11, as shown in FIG. 1A(SEQ ID NO:2).

In general, naturally occurring allelic variants of human 22P4F11 willshare a high degree of structural identity and homology (e.g., 90% ormore identity). Typically, allelic variants of the 22P4F11 proteins willcontain conservative amino acid substitutions within the 22P4F11sequences described herein or will contain a substitution of an aminoacid from a corresponding position in a 22P4F11 homologue. One class of22P4F11 allelic variants will be proteins that share a high degree ofhomology with at least a small region of a particular 22P4F11 amino acidsequence, but will further contain a radical departure form thesequence, such as a non-conservative substitution, truncation, insertionor frame shift.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

22P4F11 proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the 22P4F11protein from cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated 22P4F11 protein. A purified 22P4F11protein molecule will be substantially free of other proteins ormolecules which impair the binding of 22P4F11 to antibody or otherligand. The nature and degree of isolation and purification will dependon the intended use. Embodiments of a 22P4F11 protein include a purified22P4F11 protein and a functional, soluble 22P4F11 protein. In one form,such functional, soluble 22P4F11 proteins or fragments thereof retainthe ability to bind antibody or other ligand.

The invention also provides 22P4F11 polypeptides comprising biologicallyactive fragments of the 22P4F11 amino acid sequence, such as apolypeptide corresponding to part of the amino acid sequences for22P4F11 as shown in FIG. 1A (SEQ ID NO:2). Such polypeptides of theinvention exhibit properties of the 22P4F11 protein, such as the abilityto elicit the generation of antibodies which specifically bind anepitope associated with the 22P4F11 protein.

22P4F11 polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human 22P4F11 proteinsdisclosed herein. Alternatively, recombinant methods can be used togenerate nucleic acid molecules that encode a polypeptide fragment of a22P4F11 protein. In this regard, the 22P4F11-encoding nucleic acidmolecules described herein provide means for generating definedfragments of 22P4F11 proteins. 22P4F11 polypeptides are particularlyuseful in generating and characterizing domain specific antibodies(e.g., antibodies recognizing an extracellular or intracellular epitopeof a 22P4F11 protein), in identifying agents or cellular factors thatbind to 22P4F11 or a particular structural domain thereof, and invarious therapeutic contexts, including but not limited to cancervaccines.

Polypeptides comprising amino acid sequences which are unique to aparticular 22P4F11 protein (relative to other 22P4F11 proteins) may beused to generate antibodies which will specifically react with thatparticular 22P4F11 protein.

22P4F11 polypeptides containing particularly interesting structures canbe predicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-22P4F11 antibodies or in identifying cellular factors thatbind to 22P4F11.

In a specific embodiment described in the examples which follow, 22P4F11is conveniently expressed in 293T cells transfected with a CMV-drivenexpression vector encoding 22P4F11 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen). The secreted HIS-tagged 22P4F11 in theculture media may be purified using a nickel column using standardtechniques.

22P4F11 Antibodies

Another aspect of the invention provides antibodies that bind to 22P4F11proteins and polypeptides. The most preferred antibodies willspecifically bind to a 22P4F11 protein and will not bind (or will bindweakly) to non-22P4F11 proteins and polypeptides. Anti-22P4F11antibodies that are particularly contemplated include monoclonal andpolyclonal antibodies as well as fragments containing the antigenbinding domain and/or one or more complementarity determining regions ofthese antibodies. As used herein, an antibody fragment is defined as atleast a portion of the variable region of the immunoglobulin moleculewhich binds to its target, i.e., the antigen binding region.

22P4F11 antibodies of the invention may be particularly useful inprostate cancer diagnostic and prognostic assays, and imagingmethodologies. Intracellularly expressed antibodies (e.g., single chainantibodies) may be therapeutically useful in treating cancers in whichthe expression of 22P4F11 is involved, such as for example advanced andmetastatic prostate cancers. Similarly, such antibodies may be useful inthe treatment, diagnosis, and/or prognosis of other cancers, to theextent 22P4F11 is also expressed or overexpressed in other types ofcancer.

The invention also provides various immunological assays useful for thedetection and quantification of 22P4F11 and mutant 22P4F11 proteins andpolypeptides. Such assays generally comprise one or more 22P4F11antibodies capable of recognizing and binding a 22P4F11 or mutant22P4F11 protein, as appropriate, and may be performed within variousimmunological assay formats well known in the art, including but notlimited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer and other cancers expressing22P4F11 are also provided by the invention, including but limited toradioscintigraphic imaging methods using labeled 22P4F11 antibodies.Such assays may be clinically useful in the detection, monitoring, andprognosis of 22P4F11 expressing cancers such as prostate cancer.

22P4F11 antibodies may also be used in methods for purifying 22P4F11 andmutant 22P4F11 proteins and polypeptides and for isolating 22P4F11homologues and related molecules. For example, in one embodiment, themethod of purifying a 22P4F11 protein comprises incubating a 22P4F11antibody, which has been coupled to a solid matrix, with a lysate orother solution containing 22P4F11 under conditions which permit the22P4F11 antibody to bind to 22P4F11; washing the solid matrix toeliminate impurities; and eluting the 22P4F11 from the coupled antibody.Other uses of the 22P4F11 antibodies of the invention include generatinganti-idiotypic antibodies that mimic the 22P4F11 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a 22P4F11 protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 22P4F11 mayalso be used, such as a 22P4F11 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the openreading frame amino acid sequence of FIG. 1A (SEQ ID NO:2) may beproduced and used as an immunogen to generate appropriate antibodies. Inanother embodiment, a 22P4F11 peptide may be synthesized and used as animmunogen.

In addition, naked DNA immunization techniques known in the art may beused (with or without purified 22P4F11 protein or 22P4F11 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of the 22P4F11 as shown in FIG. 1A (SEQ ID NO:2)may be used to select specific regions of the 22P4F11 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the 22P4F11 amino acid sequence may be used to identifyhydrophilic regions in the 22P4F11 structure. Regions of the 22P4F11protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Methods for the generation of22P4F11 antibodies are further illustrated by way of the examplesprovided herein.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a 22P4F11 immunogen is conducted generallyby injection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

22P4F11 monoclonal antibodies are preferred and may be produced byvarious means well known in the art. For example, immortalized celllines which secrete a desired monoclonal antibody may be prepared usingthe standard hybridoma technology of Kohler and Milstein ormodifications which immortalize producing B cells, as is generallyknown. The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the 22P4F11 protein or a22P4F11 fragment. When the appropriate immortalized cell culturesecreting the desired antibody is identified, the cells may be expandedand antibodies produced either from in vitro cultures or from ascitesfluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the 22P4F11 protein can also be produced in thecontext of chimeric or CDR grafted antibodies of multiple speciesorigin. Humanized or human 22P4F11 antibodies may also be produced andare preferred for use in therapeutic contexts. Methods for humanizingmurine and other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al., 1986, Nature 321: 522-525;Riechmnan et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988,Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl.Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539).

Fully human 22P4F11 monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human 22P4F11 monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 22P4F11 antibodies with a 22P4F11 protein may beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,22P4F11 proteins, peptides, 22P4F11-expressing cells or extractsthereof.

A 22P4F11 antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more 22P4F11 epitopes may be generated using methodsgenerally known in the art. Homodimeric antibodies may also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560-2565).

Methods for the Detection of 22P4F11

Another aspect of the present invention relates to methods for detecting22P4F11 polynucleotides and 22P4F11 proteins, as well as methods foridentifying a cell which expresses 22P4F11.

More particularly, the invention provides assays for the detection of22P4F11 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 22P4F11 polynucleotides include, for example, a 22P4F11gene or fragments thereof, 22P4F11 mRNA, alternative splice variant22P4F11 mRNAs, and recombinant DNA or RNA molecules containing a 22P4F11polynucleotide. A number of methods for amplifying and/or detecting thepresence of 22P4F11 polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 22P4F11 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a22P4F11 polynucleotides as sense and antisense primers to amplify22P4F11 cDNAs therein; and detecting the presence of the amplified22P4F11 cDNA. In another embodiment, a method of detecting a 22P4F11gene in a biological sample comprises first isolating genomic DNA fromthe sample; amplifying the isolated genomic DNA using 22P4F11polynucleotides as sense and antisense primers to amplify the 22P4F11gene therein; and detecting the presence of the amplified 22P4F11 gene.Any number of appropriate sense and antisense probe combinations may bedesigned from the nucleotide sequences provided for the 22P4F11 (FIGS. 1and 2) (SEQ ID NOS: 1 and 3) and used for this purpose.

The invention also provides assays for detecting the presence of a22P4F11 protein in a tissue of other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting a 22P4F11 protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western Blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, in one embodiment, a method of detecting the presenceof a 22P4F11 protein in a biological sample comprises first contactingthe sample with a 22P4F11 antibody, a 22P4F11-reactive fragment thereof,or a recombinant protein containing an antigen binding region of a22P4F11 antibody; and then detecting the binding of 22P4F11 protein inthe sample thereto.

Methods for identifying a cell which expresses 22P4F11 are alsoprovided. In one embodiment, an assay for identifying a cell whichexpresses a 22P4F11 gene comprises detecting the presence of 22P4F11mRNA in the cell. Methods for the detection of particular mRNAs in cellsare well known and include, for example, hybridization assays usingcomplementary DNA probes (such as in situ hybridization using labeled22P4F11 riboprobes, Northern blot and related techniques) and variousnucleic acid amplification assays (such as RT-PCR using complementaryprimers specific for 22P4F11, and other amplification type detectionmethods, such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell which expresses a 22P4F11gene comprises detecting the presence of 22P4F11 protein in the cell orsecreted by the cell. Various methods for the detection of proteins arewell known in the art and may be employed for the detection of 22P4F11proteins and 22P4F11 expressing cells.

22P4F11 expression analysis may also be useful as a tool for identifyingand evaluating agents which modulate 22P4F11 gene expression. Forexample, 22P4F11 expression is significantly upregulated in prostatecancer, and may also be expressed in other cancers. Identification of amolecule or biological agent that could inhibit 22P4F11 expression orover-expression in cancer cells may be of therapeutic value. Such anagent may be identified by using a screen that quantifies 22P4F11expression by RT-PCR, nucleic acid hybridization or antibody binding.

Assays for Determining 22P4F11 Expression Status

Determining the status of 22P4F11 expression patterns in an individualmay be used to diagnose cancer and may provide prognostic informationuseful in defining appropriate therapeutic options. Similarly, theexpression status of 22P4F11 may provide information useful forpredicting susceptibility to particular disease stages, progression,and/or tumor aggressiveness. The invention provides methods and assaysfor determining 22P4F11 expression status and diagnosing cancers whichexpress 22P4F11, such as cancers of the prostate. 22P4F11 expressionstatus in patient samples may be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, western blot analysis of clinical samples andcell lines, and tissue array analysis.

In one aspect, the invention provides assays useful in determining thepresence of cancer in an individual, comprising detecting a significantincrease in 22P4F11 mRNA or protein expression in a test cell or tissuesample relative to expression levels in the corresponding normal cell ortissue. The presence of 22P4F11 mRNA may, for example, be evaluated intissue samples including but not limited to colon, lung, prostate,pancreas, bladder, breast, ovary, cervix, testis, head and neck, brain,stomach, etc. The presence of significant 22P4F11 expression in any ofthese tissues may be useful to indicate the emergence, presence and/orseverity of these cancers, since the corresponding normal tissues do notexpress 22P4F11 mRNA or express it at lower levels.

In a related embodiment, 22P4F11 expression status may be determined atthe protein level rather than at the nucleic acid level. For example,such a method or assay would comprise determining the level of 22P4F11protein expressed by cells in a test tissue sample and comparing thelevel so determined to the level of 22P4F11 expressed in a correspondingnormal sample. In one embodiment, the presence of 22P4F11 protein isevaluated, for example, using immunohistochemical methods. 22P4F11antibodies or binding partners capable of detecting 22P4F11 proteinexpression may be used in a variety of assay formats well known in theart for this purpose.

In addition, peripheral blood may be conveniently assayed for thepresence of cancer cells, including but not limited to prostate cancers,using RT-PCR to detect 22P4F11 expression. The presence of RT-PCRamplifiable 22P4F11 mRNA provides an indication of the presence of thecancer. RT-PCR detection assays for tumor cells in peripheral blood arecurrently being evaluated for use in the diagnosis and management of anumber of human solid tumors. In the prostate cancer field, theseinclude RT-PCR assays for the detection of cells expressing PSA and PSM(Verkaik et al., 1997, Urol. Res. 25: 373-384; Ghossein et al., 1995, J.Clin. Oncol. 13: 1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688). RT-PCR assays are well known in the art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detecting22P4F11 mRNA or 22P4F11 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 22P4F11 mRNAexpression present is proportional to the degree of susceptibility. In aspecific embodiment, the presence of 22P4F11 in prostate tissue isexamined, with the presence of 22P4F11 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor).

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of 22P4F11mRNA or 22P4F11 protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of 22P4F11 mRNA or22P4F11 protein expressed in a corresponding normal tissue taken fromthe same individual or a normal tissue reference sample, wherein thedegree of 22P4F11 mRNA or 22P4F11 protein expression in the tumor samplerelative to the normal sample indicates the degree of aggressiveness. Ina specific embodiment, aggressiveness of prostate tumors is evaluated bydetermining the extent to which 22P4F11 is expressed in the tumor cells,with higher expression levels indicating more aggressive tumors.

Methods for detecting and quantifying the expression of 22P4F11 mRNA orprotein are described herein and use standard nucleic acid and proteindetection and quantification technologies well known in the art.Standard methods for the detection and quantification of 22P4F11 mRNAinclude in situ hybridization using labeled 22P4F11 riboprobes, Northernblot and related techniques using 22P4F11 polynucleotide probes, RT-PCRanalysis using primers specific for 22P4F11, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR may beused to detect and quantify 22P4F11 mRNA expression as described in theExamples which follow. Any number of primers capable of amplifying22P4F11 may be used for this purpose, including but not limited to thevarious primer sets specifically described herein. Standard methods forthe detection and quantification of protein may be used for thispurpose. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 22P4F11 protein may be used inan immunohistochemical assay of biopsied tissue.

Therapeutic Methods and Compositions

The identification of 22P4F11 as a normally testis-specific protein thatis also expressed in cancers of the prostate (and possibly othercancers), opens a number of therapeutic approaches to the treatment ofsuch cancers.

Accordingly, therapeutic approaches aimed at inhibiting the activity ofthe 22P4F11 protein are expected to be useful for patients sufferingfrom prostate cancer and other cancers expressing 22P4F11. Thesetherapeutic approaches generally fall into two classes. In this regard,a variety of methods for inhibiting the transcription of the 22P4F11gene or translation of 22P4F11 mRNA may be employed.

A. Therapeutic Inhibition of 22P4F11 with Intracellular Antibodies

Recombinant vectors encoding single chain antibodies which specificallybind to 22P4F11 may be introduced into 22P4F11 expressing cells via genetransfer technologies, wherein the encoded single chain anti-22P4F11antibody is expressed intracellularly, binds to 22P4F11 protein, andthereby inhibits its function. Methods for engineering suchintracellular single chain antibodies are well known. Such intracellularantibodies, also known as “intrabodies”, may be specifically targeted toa particular compartment within the cell, providing control over wherethe inhibitory activity of the treatment will be focused. Thistechnology has been successfully applied in the art (for review, seeRichardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have beenshown to virtually eliminate the expression of otherwise abundant cellsurface receptors. See, for example, Richardson et al., 1995, Proc.Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem.289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies may beexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals maybe engineered into recombinant polynucleotide vectors encoding suchsingle chain antibodies in order to precisely target the expressedintrabody to the desired intracellular compartment. For example,intrabodies targeted to the endoplasmic reticulum (ER) may be engineeredto incorporate a leader peptide and, optionally, a C-terminal ERretention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus may be engineered to include anuclear localization signal. Lipid moieties may be joined to intrabodiesin order to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

In order to specifically direct the expression of such intrabodies toparticular tumor cells, the transcription of the intrabody may be placedunder the regulatory control of an appropriate tumor-specific promoterand/or enhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer may beutilized (See, for example, U.S. Pat. No. 5,919,652).

B. Therapeutic Methods Based on Inhibition of 22P4F11 Transcription orTranslation

Also provided are various methods and compositions for inhibiting thetranscription of the 22P4F11 gene. Similarly, the invention alsoprovides methods and compositions for inhibiting the translation of22P4F11 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 22P4F11gene comprises contacting the 22P4F11 gene with a 22P4F11 antisensepolynucleotide. In another approach, a method of inhibiting 22P4F11 mRNAtranslation comprises contacting the 22P4F11 mRNA with an antisensepolynucleotide. In another approach, a 22P4F11 specific ribozyme may beused to cleave the 22P4F11 message, thereby inhibiting translation. Suchantisense and ribozyme based methods may also be directed to theregulatory regions of the 22P4F11 gene, such as the 22P4F11 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a22P4F11 gene transcription factor may be used to inhibit 22P4F11 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors which inhibit the transcription of 22P4F11 throughinterfering with 22P4F11 transcriptional activation may also be usefulfor the treatment of cancers expressing 22P4F11. Similarly, factorswhich are capable of interfering with 22P4F11 processing may be usefulfor the treatment of cancers expressing 22P4F11. Cancer treatmentmethods utilizing such factors are also within the scope of theinvention.

C. General Considerations

Gene transfer and gene therapy technologies may be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizing 22P4F11(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 22P4F11 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 22P4F11 antisensepolynucleotides, ribozymes, factors capable of interfering with 22P4F11transcription, and so forth, may be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches may be combined with chemotherapy orradiation therapy regimens. These therapeutic approaches may also enablethe use of reduced dosages of chemotherapy and/or less frequentadministration, particularly in patients that do not tolerate thetoxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, may beevaluated using various in vitro and in vivo assay systems. In vitroassays for evaluating therapeutic potential include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 22P4F11 to a bindingpartner, etc.

In vivo, the effect of a 22P4F11 therapeutic composition may beevaluated in a suitable animal model. For example, xenogenic prostatecancer models wherein human prostate cancer explants or passagedxenograft tissues are introduced into immune compromised animals, suchas nude or SCID mice, are appropriate in relation to prostate cancer andhave been described (Klein et al., 1997, Nature Medicine 3: 402-408).For Example, PCT Patent Application WO98/16628, Sawyers et al.,published Apr. 23, 1998, describes various xenograft models of humanprostate cancer capable of recapitulating the development of primarytumors, micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays which measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

In vivo assays which qualify the promotion of apoptosis may also beuseful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to un-treated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material which when combined with the therapeuticcomposition retains the anti-tumor function of the therapeuticcomposition and is non-reactive with the patient's immune system.Examples include, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations may be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancerand will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

The invention further provides prostate cancer vaccines comprising a22P4F11 protein or fragment thereof, as well as DNA based vaccines. Inview of the testis-restricted expression of 22P4F11 in normal humantissues (and the existence of the testis-blood barrier), 22P4F11 cancervaccines are expected to be effective at specifically preventing and/ortreating 22P4F11 expressing cancers without creating non-specificeffects on non-target tissues. The use of a tumor antigen in a vaccinefor generating humoral and cell-mediated immunity for use in anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995,Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159:3113-3117). Such methods can be readily practiced by employing a 22P4F11protein, or fragment thereof, or a 22P4F11-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the 22P4F11 immunogen.

For example, viral gene delivery systems may be used to deliver a22P4F11-encoding nucleic acid molecule. Various viral gene deliverysystems which can be used in the practice of this aspect of theinvention include, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a 22P4F11 protein or fragment thereof introduced into thepatient (e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human 22P4F11 cDNA may be employed. Inanother embodiment, 22P4F11 nucleic acid molecules encoding specificcytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes canbe determined using specific algorithms (e.g., Epimer, Brown University)to identify peptides within a 22P4F11 protein which are capable ofoptimally binding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present 22P4F11 antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al.,1996, Prostate 29: 371-380). Dendritic cells can be used to present22P4F11 peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed with22P4F11 peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete 22P4F11protein. Yet another embodiment involves engineering the overexpressionof the 22P4F11 gene in dendritic cells using various implementingvectors known in the art, such as adenovirus (Arthur et al., 1997,Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al., 1996, CancerRes. 56: 3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869), andtumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells expressing 22P4F11 may also be engineered to expressimmune modulators, such as GM-CSF, and used as immunizing agents.

Anti-idiotypic anti-22P4F11 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 22P4F11 protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-22P4F11 antibodies that mimic an epitope ona 22P4F11 protein (see, for example, Wagner et al., 1997, Hybridoma 16:33-40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al.,1996, Cancer Immunol Immunother 43: 65-76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing 22P4F11. Constructs comprising DNA encoding a22P4F11 protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded 22P4F11 protein/immunogen. Expression of the 22P4F11 proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against prostate cancer. Variousprophylactic and therapeutic genetic immunization techniques known inthe art may be used.

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe which is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a 22P4F11protein or a 22P4F11 gene or message, respectively. Where the kitutilizes nucleic acid hybridization to detect the target nucleic acid,the kit may also have containers containing nucleotide(s) foramplification of the target nucleic acid sequence and/or a containercomprising a reporter-means, such as a biotin-binding protein, such asavidin or streptavidin, bound to a reporter molecule, such as anenzymatic, florescent, or radioisotope label.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples which follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the 22P4F11 GeneMaterials and Methods LAPC Xenografts:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408).Androgen dependent and independent LAPC-4 xenografts LAPC-4 AD and AI,respectively) and LAPC-9 AD xenografts were grown in male SCID mice andwere passaged as small tissue chunks in recipient males. LAPC-4 AIxenografts were derived from LAPC-4 AD tumors. Male mice bearing LAPC-4AD tumors were castrated and maintained for 2-3 months. After the LAPC-4tumors re-grew, the tumors were harvested and passaged in castratedmales or in female SCID mice.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/108 cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 5) 5′TTTTGATCAAGCTT₃₀3′Adaptor 1: (SEQ ID NO: 6) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′3′GGCCCGTCCTAG5′ (SEQ ID NO: 15) Adaptor 2: (SEQ ID NO: 7)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ 3′CGGCTCCTAG5′ (SEQ ID NO:16) PCR primer 1: (SEQ ID NO: 8) 5′CTAATACGACTCACTATAGGGC3′ Nestedprimer (NP)1: (SEQ ID NO: 9) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer(NP)2: (SEQ ID NO: 10) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes which may be differentially expressed in prostatecancer.

Double stranded cDNAs corresponding to the LAPC-4 AI xenograft (tester)and the LAPC-4 AD xenograft (driver) were synthesized from 2 μg ofpoly(A)+RNA isolated from xenograft tissue, as described above, usingCLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotideDPNCDN as primer. First- and second-strand synthesis were carried out asdescribed in the Kit's user manual protocol (CLONTECH Protocol No.PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with DpnII for 3 hrs. at 37° C. Digested cDNA was extracted withphenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA (LAPC-4 AD) was generated by combining in a 1:1 ratio Dpn IIdigested LAPC-4 AD cDNA with a mix of digested cDNAs derived from humanbenign prostatic hyperplasia (BPH), the human cell lines HeLa, 293,A431, Colo205, and mouse liver.

Tester cDNA (LAPC-4 AI) was generated by diluting 1 μl of Dpn IIdigested LAPC-4 AI cDNA (400 ng) in 5 μl of water. The diluted cDNA (2μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM),in separate ligation reactions, in a total volume of 10 μl at 16° C.overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 pt (600 ng) ofdriver cDNA to each of two tubes containing 1.5 pt (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 pt, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 pt of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 pt of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 pt of thediluted final hybridization mix was added to 1 pt of PCR primer 1 (10μM), 0.5 pt dNTP mix (10 μM), 2.5 pt 10× reaction buffer (CLONTECH) and0.5 pt 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 pt. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 pt from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12 18priming using the Gibco BRL Superscript Preamplification system. Themanufacturers protocol was used and included an incubation for 50 min at42° C. with reverse transcriptase followed by RNAse H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 pt with water prior to normalization. First strand cDNAs from 16different normal human tissues were obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO:13) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO:14) to amplifyβ-actin. First strand cDNA (5 pt) was amplified in a total volume of 50pt containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five pt of the PCR reaction was removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: initial denaturation was at 94° C. for 15 sec, followed by a18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 bp β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization were required to achieve equal band intensitiesin all tissues after 22 cycles of PCR.

To determine expression levels of the 22P4F11 gene, 5 pt of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs, which were designed withthe assistance of MIT:

(SEQ ID NO: 1) 5′- GCC ACA AAC AGA ATG CAA TGA AAG -3′ (SEQ ID NO: 12)5′- AAA CTG CCT GTG GTG AAA GTA GA -3′

Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results:

The SSH experiment described in the Materials and Methods, supra, led tothe isolation of numerous candidate gene fragment clones (SSH clones).All candidate clones were sequenced and subjected to homology analysisagainst all sequences in the major public gene and EST databases inorder to provide information on the identity of the corresponding geneand to help guide the decision to analyze a particular gene fordifferential expression. In general, gene fragments which had nohomology to any known sequence in any of the searched databases, andthus considered to represent novel genes, as well as gene fragmentsshowing homology to previously sequenced expressed sequence tags (ESTs),were subjected to differential expression analysis by RT-PCR and/orNorthern analysis.

One of the SHH clones comprising about 209 bp, showed no homology to anyknown gene, and was designated 22P4F11. The nucleotide sequence of thisSHH clone is shown in FIG. 1B (SEQ ID NO:3). This partial cDNA sequenceof the 22P4F11 gene encodes an open reading frame of 69 amino acids.Differential expression analysis by RT-PCR showed expression in all LAPCxenografts and in normal prostate at approximately equal levels (FIG. 2,Panel A). In addition, further RT-PCR expression analysis of firststrand cDNAs from 16 normal tissues detected high level expression ofthe 22P4F11 gene only in prostate, testis, lung and liver (FIG. 2,panels B and C).

Example 2 Isolation of Full Length cDNA Encoding the 22P4F11 Gene

The 22P4F11 SSH fragment (Example 1) was used to isolate additionalcDNAs encoding this gene. Briefly, a normal human prostate cDNA library(Clontech) was screened with a labeled probe generated from the 22P4F11cDNA. One of the positive clones, clone GTP3E10, was 2250 bp in length,and encoded the entire open reading frame of the 22P4F11 gene. The22P4F11-GTP3E10 cDNA encodes a 387 amino acid protein and contains apredicted mitochondrial signal sequence. The 22P4F11-GTP3E10 cDNA wasdeposited as plasmid p22P4F11-GTP3E10 with the American Type CultureCollection (ATCC) on Nov. 11, 1998 as has been accorded ATCC AccessionNumber 98985. The nucleotide and deduced amino acid sequences of the22P4F11 gene encoded by this cDNA are shown in FIG. 1A (SEQ ID NOS: 1and 2, respectively).

Example 3 Northern Blot Analysis of 22P4F11 RNA Expression

22P4F11 mRNA expression in normal human tissues was conducted byNorthern blotting two multiple tissue blots obtained from Clontech (PaloAlto, Calif.), comprising a total of 16 different normal human tissues,using labeled 22P4F11-GTP3E10 cDNA as a probe. RNA samples werequantitatively normalized with a β-actin probe. The results are shown inFIGS. 3A&B. Expression was only detected in testis.

To analyze 22P4F11 expression in human cancer tissues and cell lines,RNAs derived from human prostate cancer xenografts, normal prostate anda prostate cancer cell line were also analyzed. The results are shown inFIG. 3C. Strong expression was detected in one of the LAPC-4 xenografts,and lower level expression was detected in the LNCaP prostate cancercell line and in some of the other xenografts. No message was detectedin normal prostate.

Example 4 Generation of 22P4F11 Polyclonal Antibodies

To generate polyclonal antibodies directed against 22P4F11 a peptidecorresponding to a predicted antigenic sequence is designed from acoding region of the 22P4F11 protein (FIG. 1A) (SEQ ID NO:2). Thepeptide is then conjugated to keyhole limpet hemocyanin (KLH) and wasused to immunize a rabbit.

To test the rabbit serum for reactivity with 22P4F11 proteins, fulllength 22P4F11 and 22P4F11 cDNAs are cloned into an expression vectorthat provides a 6H is tag at the carboxyl-terminus (pCDNA 3.1 myc-his,InVitrogen). After transfection of the constructs into 293T cells, celllysates are probed with anti-His antibody (Santa Cruz) and theanti-22P4F11 serum using Western blotting.

Example 5 Production of Recombinant 22P4F11 in a Mammalian Systems

To express recombinant 22P4F11, the full length 22P4F11 cDNA is clonedinto an expression vector that provides a 6H is tag at thecarboxyl-terminus (pCDNA 3.1 myc-his, InVitrogen). The construct istransfected into 293T cells. Transfected 293T cell lysates are probedwith anti-22P4F11 polyclonal serum prepared, as described in Example 5above, in a Western blot.

The 22P4F11 genes are subcloned into the retroviral expression vectorpSRαMSVtkneo and used to establish 22P4F11 expressing cell lines asfollows. The 22P4F11 coding sequence (from translation initiation ATG tothe termination codons) is amplified by PCR using ds cDNA template from22P4F11 cDNA. The PCR product is subcloned into pSRαMSVtkneo via theEcoR1 (blunt-ended) and Xba 1 restriction sites on the vector andtransformed into DH5α competent cells. Colonies are picked to screen forclones with unique internal restriction sites on the cDNA. The positiveclone is confirmed by sequencing of the cDNA insert. Retroviruses areused for infection and generation of various cell lines using, forexample, 3T3CL7, PC3, and LnCap cells.

Example 6 Production of Recombinant 22P4F11 in a Baculovirus System

To generate a recombinant 22P4F11 protein in a baculovirus expressionsystem, the 22P4F11 cDNA is cloned into the baculovirus transfer vectorpBlueBac 4.5 (Invitrogen) which provides a His-tag at the N-terminusSpecifically, pBlueBac-22P4F11 is co-transfected with helper plasmidpBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cellsto generate recombinant baculovirus (see Invitrogen instruction manualfor details). Baculovirus is then collected from cell supernatant andpurified by plaque assay.

Recombinant 22P4F11 protein is then generated by infection of HighFiveinsect cells (InVitrogen) with the purified baculovirus. Recombinant22P4F11 protein may be detected using anti-22P4F11 antibody. 22P4F11protein may be purified and used in various cell based assays or asimmunogen to generate polyclonal and monoclonal antibodies specific for22P4F11.

Example 7 Identification of Potential Signal Transduction Pathways

To determine whether 22P4F11 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressing22P4F11. These transcriptional reporters contain consensus binding sitesfor known transcription factors which lie downstream of wellcharacterized signal transduction pathways. The reporters and examplesof there associated transcription factors, signal transduction pathways,and activation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

2P4F11-mediated effects may be assayed in cells showing mRNA expression.Luciferase reporter plasmids may be introduced by lipid mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellsextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Example 8 Generation of 22P4F11 Monoclonal Antibodies

In order to generate 22P4F11 monoclonal antibodies, aglutathione-S-transferase (GST) fusion protein encompassing a 22P4F11protein is synthesized and used as immunogen. Balb C mice are initiallyimmunized intraperitoneally with 200 □g of the GST-22P4F11 fusionprotein mixed in complete Freund's adjuvant. Mice are subsequentlyimmunized every 2 weeks with 75 μg of GST-22P4F11 protein mixed inFreund's incomplete adjuvant for a total of 3 immunizations. Reactivityof serum from immunized mice to full length 22P4F11 protein isconveniently monitored by ELISA using a partially purified preparationof HIS-tagged 22P4F11 protein expressed from 293T cells. Mice showingthe strongest reactivity are rested for 3 weeks and given a finalinjection of fusion protein in PBS and then sacrificed 4 days later. Thespleens of the sacrificed mice are then harvested and fused to SPO/2myeloma cells using standard procedures (Harlow and Lane, 1988).Supernatants from growth wells following HAT selection are screened byELISA and Western blot to identify 22P4F11 specific antibody producingclones.

The binding affinity of a 22P4F11 monoclonal antibody may be determinedusing standard technology. Affinity measurements quantify the strengthof antibody to epitope binding and may be used to help define which22P4F11 monoclonal antibodies are preferred for diagnostic ortherapeutic use. The BIAcore system (Uppsala, Sweden) is a preferredmethod for determining binding affinity. The BIAcore system uses surfaceplasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Mortonand Myszka, 1998, Methods in Enzymology 295: 268) to monitorbiomolecular interactions in real time. BIAcore analysis convenientlygenerates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants.

Example 9 In Vitro Assays of 22P4F11 Function

The expression of 22P4F11 in prostate cancer suggests a possiblefunctional role in tumor progression. 22P4F11 function can be assessedin mammalian cells using in vitro approaches. For mammalian expression,22P4F11 can be cloned into a number of appropriate vectors, includingpcDNA 3.1 myc-His-tag (Example 6) and the retroviral vector pSRαtkneo(Muller et al., 1991, MCB 11:1785). Using such expression vectors,22P4F11 can be expressed in several cell lines, including PC-3, NIH 3T3,LNCaP and 293T. Expression of 22P4F11 can be monitored usinganti-22P4F11 antibodies.

Mammalian cell lines expressing 22P4F11 can be tested in several invitro and in vivo assays, including cell proliferation in tissueculture, activation of apoptotic signals, tumor formation in SCID mice,and in vitro invasion using a membrane invasion culture system (MICS)(Welch et al., Int. J. Cancer 43: 449-457). 22P4F11 cell phenotype iscompared to the phenotype of cells that lack expression of 22P4F11.

Cell lines expressing 22P4F11 can also be assayed for alteration ofinvasive and migratory properties by measuring passage of cells througha matrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and 22P4F11 overexpressing PC3, 3T3 and LNCaPcells. To assay whether 22P4F11 has chemoattractant properties, parentalindicator cells are monitored for passage through the porous membranetoward a gradient of 22P4F11 conditioned media compared to controlmedia. This assay may also be used to qualify and quantify specificneutralization of the 22P4F11 induced effect by candidate cancertherapeutic compositions.

Example 10 In Vivo Assay for 22P4F11 Tumor Growth Promotion

The effect of the 22P4F11 protein on tumor cell growth may be evaluatedin vivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected SQ on each flank with 1×106 of either PC3, TSUPR1,or DU145 cells containing tkNeo empty vector or 22P4F11. At least twostrategies may be used: (1) Constitutive 22P4F11 expression underregulation of an LTR promoter, and (2) Regulated expression undercontrol of an inducible vector system, such as ecdysone, tet, etc. Tumorvolume is then monitored at the appearance of palpable tumors andfollowed over time to determine if 22P4F11 expressing cells grow at afaster rate. Additionally, mice may be implanted with 1×105 of the samecells orthotopically to determine if 22P4F11 has an effect on localgrowth in the prostate or on the ability of the cells to metastasize,specifically to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 22P4F11 inhibitory effect ofcandidate therapeutic compositions, such as for example, 22P4F11intrabodies, 22P4F11 antisense molecules and ribozymes.

1. An antibody or fragment thereof that specifically binds to apolypeptide encoded by a polynucleotide selected from the groupconsisting of (a) a polynucleotide having the sequence of SEQ ID NO: 1;(b) a polynucleotide having the sequence of SEQ ID NO: 1, fromnucleotide residue number 306 through nucleotide residue number 1466;(c) a polynucleotide encoded by the cDNA contained in the plasmiddeposited with American Type Culture Collection as Accession Nos. 98985;and (d) a polynucleotide encoding a 22P4F11 protein having the aminoacid sequence SEQ ID NO:
 2. 2. The antibody or fragment of claim 1,wherein the polypeptide is encoded by a polynucleotide having thesequence of SEQ ID NO:
 1. 3. The antibody or fragment of claim 1,wherein the polypeptide is encoded by a polynucleotide having thesequence of SEQ ID NO: 1, from nucleotide residue number 306 throughnucleotide residue number
 1466. 4. The antibody or fragment of claim 1,wherein the polypeptide is encoded by a polynucleotide encoded by thecDNA contained in the plasmid deposited with American Type CultureCollection as Accession Nos.
 98985. 5. An antibody or fragment thereofthat specifically binds to a polypeptide having the amino acid sequenceshown in SEQ ID NO:
 2. 6. The antibody or fragment of claim 1, whereinthe antibody is a polyclonal antibody
 7. The antibody or fragment ofclaim 1, wherein the antibody is a human antibody, a humanized antibody,or a chimeric antibody.
 8. The antibody or fragment of claim 1, whereinthe antibody is a monoclonal antibody.
 9. The monoclonal antibody orfragment of claim 8, wherein the monoclonal antibody is recombinantlyproduced.
 10. The antibody or fragment of claim 9, wherein therecombinant protein comprises the antigen binding region.
 11. Themonoclonal antibody or fragment of claim 8, wherein the fragment thereofis selected from the group consisting of Fab, F(ab′)2, Fv and sFvfragment.
 12. A hybridoma that produces the monoclonal antibody of claim8.
 13. A pharmaceutical composition that comprises the antibody orfragment thereof of claim 1 and a physiologically acceptable carrier.14. The antibody or fragment of claim 1, further labeled with adetectable marker.
 15. The antibody or fragment of claim 14, wherein thedetectable marker is selected from the group consisting of aradioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator and an enzyme.
 16. A singlechain monoclonal antibody that comprises the variable domains of theheavy and light chains of a monoclonal antibody of claim
 8. 17. A vectorcomprising a polynucleotide that encodes a single chain monoclonalantibody of claim 16 that immunospecifically binds to a protein of SEQID NO:2.
 18. A pharmaceutical composition that comprises apolynucleotide that encodes a single chain monoclonal antibody thatimmunospecifically binds to a protein having an amino acid sequence asshown in SEQ ID NO:2 and a physiologically acceptable carrier.
 19. Amethod for detecting a protein comprising the amino acid sequence of SEQID NO:2 in a biological sample, comprising steps of: providing abiological sample; contacting the biological sample with the antibody ofclaim 1 that specifically binds to the protein of SEQ ID NO:2; anddetecting the presence of a complex between the protein and theantibody.
 20. A method of inhibiting in a patient the development of acancer that expresses the protein of SEQ ID NO:2, the method comprising:administering to the patient an effective amount of the composition ofclaim 18.